Cellulose is an essential constitutive component of cells of higher plants, and widely exists in nature. Cellulose is a polysaccharide polymer of glucose units polymerized through a β-1,4-glycosidic bond. In nature, cellulose exists in a crystalline or amorphous state. By bonding to other components such as lignin, hemicelluloses, and pectins in a complicated manner, cellulose constructs plant tissues.
Cellulase is a generic term of a group of enzymes that breaks down cellulose. Generally, cellulase produced by microorganisms includes various types of cellulase components. In accordance with the substrate specificity, the cellulase components are classified into three types: cellobiohydrolase, endoglucanase, and β-glucosidase. Aspergillus niger that is a cellulase-producing filamentous fungus is believed to produce 4 types of cellobiohydrolases at maximum, 15 types of endoglucanases, and 15 types of β-glucosidases. Presumably, multiple enzymes among these acting in various reaction modes compensate for each other to exhibit synergistic effects, thereby breaking down cellulose which is an essential component of plant cell walls. It is believed that β-glucosidase catalyzes a reaction to release glucose from cello-oligosaccharides, cellobiose or glycosides with aglycone linked thereto through β-D-glucopyranosyl linkage. β-glucosidase is an important enzyme in the final stage of cellulose saccharification and in releasing glucose from glycoside.
Ethanol conversion from biomass has advantages that: the raw material is more readily available, combustion of the raw material or burying in the ground can be avoided, and ethanol fuel is environmentally clean. Woods, agricultural residues, herbaceous crops and municipal solid wastes have drawn attention as biomass for ethanol production. These materials are mainly composed of cellulose, hemicellulose and lignin. Once cellulose is converted into glucose, the glucose is easily fermented into ethanol by yeasts. On the other hand, cellobiose is not easily fermented into ethanol by yeasts, and accordingly the remaining cellobiose causes ethanol yield reduction. What is more important is that cellobiose is a potent inhibitor of endoglucanases and cellobiohydrolases. For this reason, the accumulation of cellobiose during hydrolysis is not desirable for production of ethanol. Generally, cellulase-producing microorganisms can hardly produce β-glucosidases. This brings about a major problem that cellobiose produced by enzymatic hydrolysis is accumulated.
In order to promote conversion from cellobiose to glucoses, the yield of β-glucosidases is increased by over-expression of β-glucosidases in a host. Thus, it is effective means for promoting saccharification from biomass to glucose. Accordingly, isolation of a novel β-glucosidase gene which is introduced and expressed in cellulase-producing microorganisms has been desired.
Meanwhile, filamentous fungus Acremonium cellulolyticus has been reported to produce a cellulase having a strong saccharification power (Non Patent Literature 1) and to be highly useful for feed and silage usages (Patent Literatures 1 to 3). In addition, the cellulase component contained therein (Patent Literatures 4 to 10) has also been examined in detail. It has been revealed that various kinds of cellulase component are secreted as in other filamentous fungi. Particularly, as to the β-glucosidase activity of the cellulase therein, it has been reported, for example, that the activity is significantly higher than those of cellulases from Trichoderma reesei and the like (Patent Literature 11). Because of such characteristics, attention has been focused on Acremonium cellulolyticus as the target from which a β-glucosidase gene is isolated.
However, only a few genes have been isolated from Acremonium cellulolyticus so far (Patent Literatures 9, 10). Further, the isolated genes have not yet been successfully expressed in filamentous fungi other than Acremonium cellulolyticus. 